Affinity binding-based system for detecting particulates in a fluid

• US 6,143,247 A
• Filed: 12/19/1997
• Issued: 11/07/2000
• Est. Priority Date: 12/20/1996
• Status: Expired due to Fees

• Current Assignee: Tecan Trading AG (Tecan Group AG)
• Original Assignee: Gamera Bioscience Corporation (Tecan Group AG)
• Inventors: Sheppard, Norman F. Jr., Carvalho, Bruce L., Mian, Alec, Kellogg, Gregory, Kieffer-Higgins, Stephen G.
• Primary Examiner(s): Chin, Christopher L.
• Assistant Examiner(s): Do, Pensee T.
• Application Number:
  US08/995,056
• Time in Patent Office: 1,054 Days
• Field of Search: 422/50, 422/101, 422/102, 422/72, 422/63, 422/64, 422/68.1, 436/45, 436/63, 436/177, 436/180, 210/787, 210/374, 210/380.1, 210/780, 210/109, 210/781
• US Class Current: 422/63
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: B01F 33/30 Micromixers
• CPC Class Codes: B01F 35/71725 using centrifugal forces
• CPC Class Codes: B01L 2200/0605 Metering of fluids
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/021 Identification, e.g. bar codes
• CPC Class Codes: B01L 2300/024 Storing results with means integrated into the container
• CPC Class Codes: B01L 2300/069 Absorbents; Gels to retain a fluid
• CPC Class Codes: B01L 2300/0806 Standardised forms, e.g. compact disc [CD] format
• CPC Class Codes: B01L 2300/0864 comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
• CPC Class Codes: B01L 2300/0867 Multiple inlets and one sample wells, e.g. mixing, dilution
• CPC Class Codes: B01L 2300/087 Multiple sequential chambers
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2300/168 Specific optical properties, e.g. reflective coatings
• CPC Class Codes: B01L 2300/1805 Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
• CPC Class Codes: B01L 2300/1861 using radiation
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0661 shape memory polymer valves
• CPC Class Codes: B01L 2400/0677 phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
• CPC Class Codes: B01L 3/5027 : by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: B01L 3/502715 : characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/50273 : characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 : characterised by integrated valves microvalves F16K99/0001
• CPC Class Codes: B01L 7/00 : Heating or cooling apparatus autoclaves B01J3/04; Heat insulating devices
• CPC Class Codes: C07K 14/79 : Transferrins, e.g. lactoferrins, ovotransferrins
• CPC Class Codes: C12N 9/2462 : Lysozyme (3.2.1.17)
• CPC Class Codes: G01N 15/14 : Optical investigation techniques, e.g. flow cytometry
• CPC Class Codes: G01N 21/07 : Centrifugal type cuvettes G01N21/09 takes precedence
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: G01N 33/569 : for microorganisms, e.g. protozoa, bacteria, viruses
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92
• CPC Class Codes: G01N 35/00069 : whereby the sample substrate is of the bio-disk type, i.e. having the format of an optical disk
• CPC Class Codes: Y10S 366/03 : Micromixers: variable geometry from the pathway influences mixing/agitation of non-laminar fluid flow
• CPC Class Codes: Y10T 436/111666 : Utilizing a centrifuge or compartmented rotor
• CPC Class Codes: Y10T 436/25375 : Liberation or purification of sample or separation of material from a sample [e.g., filtering, centrifuging, etc.]
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

This invention provides methods an apparatus for detecting and quantifying particulate matter suspended in a fluid. Specifically, the invention provides an integrated, affinity-binding based, analytical system comprising a platform for performing an affinity-binding based assay for specifically binding particulates including microbial cells, and a detection means for detecting the particulates specifically bound to a defined surface or chamber comprising the platform. Methods for using the analytical systems of the invention are also provided.

Claims

1. A centripetally-motivated apparatus for detecting a cell in a fluid, the apparatus comprising

a platform, comprising a substrate having a first flat, planar surface and a second flat, planar surface opposite thereto, wherein the first surface defines a cell accumulation chamber wherein a defined amount of a fluid comprising cells is retained in the chamber for a time sufficient to permit the cells to accumulate in the chamber, and further comprises a multiplicity of microchannels embedded within the first planar surface and a sample input means, wherein the cell accumulation chamber, the sample input means and the microchannels are connected and in fluid communication, aid wherein the platform further comprises

a wash buffer reservoir containing a wash buffer in fluid communication with the cell accumulation chamber;

a fluid waste receptacle in fluid communication with the cell accumulation chamber, wherein the cell accumulation chamber is positioned radially more distal from the center of the platform than the sample input means or the wash buffer reservoir and the microchannels are arrayed between the sample input means, the cell accumulation chamber, the wash buffer reservoir and the fluid waste receptacle to provide fluid communication there between;

the platform comprising the apparatus in combination with

a device, comprising a base, a rotating means, a power supply and user interface and operations controlling means, wherein the rotating means is operatively linked to the platform and in rotational contact therewith

wherein a fluid sample comprising cells is moved from the sample input means to the cell accumulation chamber of the platform by centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels, and is incubated thereon for a time sufficient to permit the cells to accumulate in the area; and

wherein the fluid sample is replaced with the wash buffer and displaced into the fluid waste receptacle by centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels; and

wherein the wash buffer is further displaced into the fluid waste receptacle by centripetal force arising from rotational motion of tile platform for a time and a rotational velocity sufficient to move the fluid through the microchannels; and

wherein a cell in the cell accumulation chamber is detected thereupon.

2. An apparatus of claim 1 wherein the sample input means further comprises a metering fluid sample application means, wherein a metered amount of an unmetered fluid sample applied to the sample input means is retained in the metering fluid sample application means, wherein the sample input means is further in fluidic contact with an overflow reservoir, wherein fluid comprising the unmetered fluid sample in excess of the metered amount of the fluid sample that is retained by the metering sample application means is transferred to the overflow reservoir.

3. An apparatus according to claim 2 wherein the surface is modified by treatment with a non-specific cell adhesion molecule or a specific binding reagent to permit the cell to attach to the surface and be retained in the cell accumulation chamber thereby.

4. The apparatus of claim 1, wherein the first flat, planar surface and second flat, planar surface of the platform form a disk.

5. An apparatus according to claim 4 wherein the metered amount of the fluid sample is from about 10 μ

L to about 500 μ

L.

6. The apparatus of claim 1, wherein the first and second flat, planar surfaces of the platform define a centrally located aperture that is engaged to a spindle on the device, whereby rotational motion of the spindle is translated into rotational motion of the platform.

7. An apparatus according to claim 1 wherein the cell accumulation chamber further comprises a filtering means having a pore size that prevents the cells from leaving the cell accumulation chamber.

8. An apparatus according to claim 1 wherein the cell accumulation chamber further comprises a non-specific cell adhesion means that prevents the cells from leaving the cell accumulation chamber.

9. An apparatus according to claim 1 for measuring an effect of a test compound on a cell, the apparatus further comprising

a reagent reservoir containing a test compound in fluid communication with the cell accumulation chamber of the platform;

wherein an amount of a fluid sample comprising a cell is moved from the fluid sample input means to the cell accumulation chamber of the platform by centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels, and incubated thereon for a time sufficient to permit the cell to accumulate in the area; and

wherein the fluid sample is replaced with the test compound by centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels, and incubated thereon for a time sufficient for the test compound to have an effect on the cell in the cell accumulation chamber; and

wherein the effect of the test compound on the cell in the cell accumulation chamber is assayed by visual inspection and is detected thereby.

10. The apparatus of claim 9 wherein the effect is cytotoxicity and is detected by a reduction of cell viability in the presence of the test compound.

11. The apparatus of claim 9 wherein the effect is cell activation comprising binding to a cellular receptor and is detected by a difference in intracellular ion concentrations in the presence and absence of the test compound.

12. An apparatus according to claim 1, further comprising:

a reservoir comprising an amount of a detectable labeling moiety that is specific for the particulate to be detected and is in fluid communication with the area of the platform that is coated with a specific binding reagent, wherein the wash buffer is replaced by the detectable labeling moiety in the cell accumulation chamber of the platform by centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels.

13. An apparatus according to claim 12, wherein the detectable labeling moiety is a histochemical stain, a fluorescent ion indicator, a electric potential-sensitive indicator dye or an ion-sensitive indicator dye.

14. An apparatus according to claim 12 wherein the detectable labeling moiety is a immunochemical reagent.

15. An apparatus according to claim 14 wherein the immunochemical reagent is a detectably labeled antibody.

16. A method for measuring an effect of a test compound on a cell using a centripetally-motivated apparatus according to claim 15, the method comprising the steps of

applying an amount of a sample comprising a particulate to the fluid sample input means of the apparatus;

moving the fluid sample from the fluid sample input means to the cell accumulation chamber by applying centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels;

incubating the fluid sample in the cell accumulation chamber and replacing the fluid of the fluid sample in the cell accumulation chamber with the wash buffer by applying centripetal force arising from rotational notion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels, wherein the cells are retained in the cell accumulation chamber;

replacing the fluid sample with the test compound in the cell accumulation chamber by applying centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels, wherein the cells are retained in the cell accumulation chamber;

incubating the cells in the presence of the test compound for a time sufficient for the test compound to have an effect on the cell in the cell accumulation chamber;

detecting the effect of the test compound on the cell in the cell accumulation chamber.

17. An apparatus according to claim 12 wherein the detectable label is a fluorescent label.

18. An apparatus according to claim 12 wherein the detectable label is produced by an enzymatic moiety, wherein the apparatus further comprises a substrate for the enzymatic moiety wherein the substrate is converted to a detectable label by enzymatic action.

19. An apparatus according to claim 12 wherein the substrate comprises a component of the wash buffer.

20. An apparatus according to claim 12 wherein the substrate comprises a component of the reservoir containing the detectable labeling moiety.

21. An apparatus according to claim 1 wherein the substrate is a component of the wash buffer.

22. An apparatus according to claim 1 wherein the detection chamber comprises a volume from about 5 μ

L to about 1000 μ

L.

23. An apparatus according to claim 1 wherein the device further comprises a light source positioned to illuminate the platform.

24. An apparatus according to claim 23 further comprising a photodetector, wherein the photodetector is positioned to detect light from the light source transmitted through or reflected from the platform surface in the cell accumulation chamber.

25. The apparatus of claim 24, wherein the photodetector is stationary and samples the cell accumulation chamber at a frequency equal to the frequency of rotation of the platform or multiples thereof.

26. An apparatus according to claim 24 wherein a portion of the cell accumulation chamber of the platform is optically transparent, and the photodetector and the light source are positioned so that light from the light source illuminates the cell accumulation chamber and is detected by the photodetector through the optically transparent portion of the platform.

27. An apparatus according to claim 24 wherein a portion of the cell accumulation chamber comprises a reflective surface, and the photodetector and the light source are positioned so that light from the light source illuminates the cell accumulation chamber is reflected therefrom and the reflected light is detected by the photodetector.

28. The apparatus of claim 24, wherein the photodetector on the device is brought into alignment with the cell accumulation chamber on the platform by rotational motion of the platform.

29. An apparatus according to claim 1 wherein the cell accumulation chamber of the platform further comprises alternating transparent and reflective regions.

30. An apparatus according to claim 29 wherein the alternating transparent and reflective regions define a pattern in the cell accumulation chamber.

31. A method for detecting a cell in a fluid using a centripetally-motivated apparatus according to claim 1, the method comprising the steps of

applying an amount of a sample comprising a particulate to the fluid sample input means of the apparatus;

moving the fluid sample from the fluid sample input means to the cell accumulation chamber by applying centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels;

incubating the fluid sample in the cell accumulation chamber and replacing the fluid of the fluid sample in the cell accumulation chamber with the wash buffer by applying centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels, wherein the cells are retained in the cell accumulation chamber;

displacing the wash buffer into the fluid waste receptacle by applying centripetal force arising from rotational motion of the platform for a time and a rotational velocity sufficient to move the fluid through the microchannels, wherein the cells are retained in the cell accumulation chamber; and

detecting the particulate retained in the cell accumulation chamber.

32. On a platform comprising an analytical apparatus for detecting a particulate in a fluid sample, a detection chamber comprising a cell adhesion agent wherein the detection chamber has a patterned reflective surface comprising an optically transparent portion and a reflective portion wherein the transparent portion of the surface is coated with the cell adhesion agent and the reflective portion of the surface is coated with a blocking agent that prevents non-specific binding thereto.

33. The detection chamber of claim 32 wherein the reflective portion of the surface is coated with a reflective metal.

34. An apparatus according to claim 33 further comprising a multiplicity of photodetectors.

35. An apparatus according to claim 34 wherein the light reflected from the reflective regions is detected by the photodetectors and is used to position and focus a beam of light in a transparent region.

36. The detection chamber of claim 32 wherein the cell adhesion agent is a specific binding reagent.

37. An apparatus according to claim 32 further comprising a light source wherein the light source produces multiple beams of light.

38. An apparatus according to claim 32 wherein the blocking reagent is present on the surface of the platform, on a reflective region thereof.

39. An apparatus according to claim 32 wherein the specific binding reagent is present on the surface of the platform on a reflective region thereof.

40. An apparatus according to claim 32 wherein the blocking reagent is present on the surface of the platform on a transparent region thereof.